RAPD and ISSR fingerprinting in cultivated chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) and its wild progenitor <Emphasis Type="Italic">Cicer reticulatum</Emphasis> Ladizinsky

Detection of genetic relationships between 19 chickpea cultivars and five accessions of its wild progenitor Cicer reticulatum Ladizinsky were investigated by using RAPD and ISSR markers. On an average, six bands per primer were observed in RAPD analysis and 11 bands per primer in ISSR analysis. In RAPD, the wild accessions shared 77.8% polymorphic bands with chickpea cultivars, whereas they shared 79.6% polymorphic bands in ISSR analysis. In RAPD analysis 51.7% and 50.5% polymorphic bands were observed among wild accessions and chickpea cultivars, respectively. Similarly, 65.63% and 56.25% polymorphic bands were found in ISSR analysis. The dendrogram developed by pooling the data of RAPD and ISSR analysis revealed that the wild accessions and the ICCV lines showed similar pattern with the dendrogram of RAPD analysis. The ISSR analysis clearly indicated that even with six polymorphic primers, reliable estimation of genetic diversity could be obtained, while nearly 30 primers are required for RAPD. Moreover, RAPD can cause genotyping errors due to competition in the amplification of all RAPD fragments. The markers generated by ISSR and RAPD assays can provide practical information for the management of genetic resources. For the selection of good parental material in breeding programs the genetic data produced through ISSR can be used to correlate with the relationship measures based on pedigree data and morphological traits to minimize the individual inaccuracies in chickpea.     

Comparative analysis of genetic diversity using molecular and morphometric markers in Andrographis paniculata (Burm. f.) Nees

Andrographis paniculata is a medicinal plant of immense therapeutic value. The present study was aimed to elucidate its genetic diversity based on morphochemical and RAPD markers from 53 accessions belonging to 5 eco-geographic regions. Analysis of variance and D ² statistics revealed significant differences in all the metric traits and sufficient inter-cluster distances indicating considerable diversity among the accessions. The complementary approach of RAPD was used to evaluate the genetic dissimilarities among all the accessions using 6 highly polymorphic primers. The average proportion of polymorphic loci across primers was 96.28%. The molecular genetic diversity based on Shannon index per primer averaged 5.585 with values ranging from 3.08 to 8.70 indicating towards wide genetic base. RAPD based UPGMA and D ² cluster analysis also revealed that various accessions available in different eco-geographic regions might have originated from native places of wild abundance. Similarity matrices were generated for molecular markers and morphometric data to determine the degree of congruence between the two. A highly significant but low correlation (r = 0.547, P < 0.001) was obtained thus implying the correspondence between the two. The species is hermaphroditic and a habitual inbreeder. The present study yielded a typical triangular congruence between its breeding system, morphometric traits and RAPD markers thus elucidating the usefulness of complementary approaches to make diversity analysis more explanatory and purposeful for optimum genetic amelioration and effective conservation of its genotypic variability.     

Genetic variation in radish (Raphanus sativus L.) germplasm from Pakistan using morphological traits and RAPDs

Genetic diversity of 30 radish (Raphanus sativus L.) accessions was investigated at the phenotypic level with morphological characters and at the DNA level using the random amplified polymorphic DNA (RAPD) technique. Thirty-six morpho-physiological traits were recorded from seedling stage to harvest. The 31 primers used generated 202 RAPD bands, of which 158 (78.2%) were polymorphic. Multivariate procedures were used to classify the germplasm on the basis of phenotypic traits and RAPD fragments. Dendrograms were generated for the Euclidean distance from the morphological data and the Nei's genetic distance from the RAPD markers. Phenotypically, all the accessions were classified into four major groups corresponding to the different forms of cultivated radish. The morphological diversity existing within each of these groups suggested that they should be discriminated into the three botanical convarieties, sativusT (large-rooted), caudatus (pod-type) and oleifer (oilseed-type). Clustering of the accessions did not show any pattern of association between the morphological characters and the collection sites. Instead, landrace groups were associated with their morphological similarities and horticultural uses. On the other hand, the intra-specific genetic relationships of several accessions based on RAPD analysis were related primarily to their collection sites rather than to their phenotypic affinities. The level of polymorphism exhibited by the various convarieties could be exploited in genetic mapping populations to tag economically important traits. These genotypes also could serve as a useful germplasm source for root, leaf, pod and seed. This preliminary study of traditional radish landraces from Pakistan provides useful information regarding their horticultural potential.     

Characterization of under-utilized fruits by molecular markers. A case study of loquat

The usefulness of RAPD markers for genotyping a minor fruit species such as loquat has been tested in order to assess their ability for identifying accessions and to provide a set of markers suitable for use by different groups of scientists and curators. Twenty-nine polymorphic markers selected from a previous study of 33 accessions were tested in 46 new accessions added to the collection. Using the same PCR standard conditions, only 20 markers out of 29 selected in the previous study gave consistent amplifications in the new set of plant material. The rest required optimization of reaction conditions. This fact pointed out that RAPD markers were sensitive to the experimental conditions, hence a standard technique did not guarantee reproducibility. To overcome this problem markers for plant fingerprinting should be selected after comparison across accession sets. Only those markers reproducible with different sampling and checked in several sets of accessions are suitable for germplasm fingerprinting. In the present study we propose 31 RAPDs for fingerprinting loquat that accomplished these characteristics.The markers obtained were sufficient for determining origin and relationships of cultivars, for identifying synonyms and derived varieties from bud-sports. All bud sports were identical for all RAPDs selected.     

Genetic variation in cucumber (Cucumis sativus L.) as assessed by random amplified polymorphic DNA1

Isozyme and restriction fragment length polymorphisms (RFLPs) have been applied to studies of genetic relationships and germplasm management in cucumber (Cucumis sativus L.). However, isozymes identify relatively few polymorphisms, and RFLPs are technically complex, expensive, and not compatible for the high through-put required for rigorous assessment of this narrow-based germplasm. Since random amplified polymorphic DNA (RAPD) markers do not manifest such shortcomings, a study was conducted in cucumber to examine genetic relationships in diverse germplasm, assess the usefulness of RAPD markers in distinguishing elite accessions, and compare the relative effectiveness of RAPD markers to that of isozyme and RFLP markers. One hundred and eighteen C. sativus accessions were analyzed using variation at 71 RAPD loci (44 mapped and 27 unmapped). Genetic distances among accessions were estimated using the simple matching coefficient complement, and analyzed using multi-dimensional scaling. Each accession had a unique marker profile, indicating that RAPD analysis was useful in genotypic differentiation. Germplasm grouping patterns were consistent with individual accession origins, theoretical dispersal routes and discriminating morphological characters (i.e., sex expression and fruit length to diameter ratio). Although elite accessions were discriminated by RAPD profiling, their genetic distances were relatively small (between 0.01 and 0.58), indicating limited genetic diversity in this germplasm array. Assessment of a subset of the germplasm array using RAPDs resulted in genetic distance measurements more similar to published genetic distance estimates by RFLP markers (Spearman rank correlation, r_s = 0.7–0.8) than estimates by isozyme markers (r_s = 0.4). Data indicate that RAPD markers have utility for analysis of genetic diversity and germplasm management in cucumber.     

Determination of genetic variability by RAPD markers in cauliflower,cabbage and kale local cultivars from France

RAPD markers were used to analyse the genetic variability among a cole crop cultivar collection from France and to identify possible duplicate accessions for facilitating the germplasm multiplication. We surveyed 24 cauliflower, 24 cabbage and 48 kale populations using 62, 100 and 89 RAPD markers respectively on 40 seed bulk samples per accession. Genetic distances were calculated on the basis of these markers. Using the phylogenetic package PAUP we compared the genetic clusters obtained for the RAPD markers with the groups based on morphologic and agronomic data. For cauliflowers and cabbages a drastic selection by growers led to an extensive differentiation of morphologic and precocity traits related to geographic origin, which matches the grouping based on RAPD markers. Furthermore, RAPDs detected in some cases misclassification of accessions in the collection. Kales formed an homogeneous group probably due to extensive genetic exchanges leading to a continuous diversity. For the other crops, different horticultural types were separated in several subgroups by the RAPD markers suggesting different genetic origins. RAPD markers provide a rapid and informative approach to analyze the genetic variability in a collection. It can be applied to autogamous (spring cauliflower) or allogamous (winter cauliflower) crops, even in cases where the intra-population diversity is extensive (cabbages).     

Analysis of genetic diversity in a sweet potato (Ipomoea batatas L.) germplasm collection from Tanzania as revealed by AFLP

Sweet potato (Ipomoea batatas L.) is the fifth most important crop in the developing countries after rice, wheat, maize and cassava. The amplified fragment length polymorphism (AFLP) method was used to study the genetic diversity and relationships of sweet potato accessions in the germplasm collection of Sokoine University of Agriculture, Morogoro and Sugarcane Research Institute, Kibaha, Tanzania. AFLP analysis of 97 sweet potato accessions using ten primer combinations gave a total of 202 clear polymorphic bands. Each one of the 97 sweet potato accessions could be distinguished based on these primer combinations. Estimates of genetic similarities were obtained by the Dice coefficient, and a final dendrogram was constructed with the un-weight pair-group method using arithmetic average. AFLP-based genetic similarity varied from 0.388 to 0.941, with a mean of 0.709. Cluster analysis using genetic similarity divided the accessions into two main groups suggesting that there are genetic relationships among the accessions. Principal Coordinate analysis confirmed the pattern of the cluster analysis. Analysis of molecular variance revealed greater variation within regions (96.19%) than among regions (3.81%). The results from the AFLP analysis revealed a relatively low genetic diversity among the germplasm accessions and the genetic distances between regions were low. A maximally diverse subset of 13 accessions capturing 97% of the molecular markers diversity was identified. We were able to detect duplicates accessions in the germplasm collection using the highly polymorphic markers obtained by AFLP, which were found to be an efficient tool to characterize the genetic diversity and relationships of sweet potato accessions in the germplasm collection in Tanzania.     

Variation and phylogeny of the triploid cultivated potatoSolanum chaucha Juz. et Buk. based on RAPD and isozyme markers

Ninety four accessions of the cultivated triploid potatoS. chaucha were analyzed and classified in genotypic groups using 9 isozyme loci and RAPD markers disclosed by 20 arbitrary 10-mer primers. Eight isozyme loci out of nine were polymorphic. A total of 22 allozymes were analyzed but none of them were specific for any genotypic group. About half (52%) of the 102 RAPD markers scored, were polymorphic, all of them showing polymorphism among groups and rarely within groups. Eighteen RAPD markers were specific for certain genotypes. The isozyme markers showed a certain amount of intra group variation which made classification less reliable than with RAPD markers. A total of 10 triploid genetic groups were discriminated using both techniques together. A single primer was found to be sufficient to distinguish all 10 groups. All varieties of a single group are considered to have been derived from the same cross and then clonally propagated, even though there is a high amount of morphological variation within a single genotypic group due probably to somatic mutations. RAPD markers have been shown to be more reliable in the classification of triploid potato varieties than other genetic markers like isozymes, proteins and morphological traits.     

Genetic diversity of Cucurbita maxima assessed using morphological characteristics and random-amplified polymorphic DNA markers in China

Cucurbita maxima is one of the most widely cultivated Cucurbitaceae in Heilongjiang province, China. The objectives of this study were to clarify the genetic diversity of 44 accessions from different geographical origins using morphological and molecular characteristics and to compare the consistency of these morphological and molecular characteristics. Morphological characteristics showed a large qualitative variability primarily according to fruit-related traits. Twenty-eight random-amplified polymorphic DNA (RAPD) markers produced 128 bands. Both morphological characteristics and RAPD markers grouped 44 accessions of C. maxima into four clusters, and the similarity coefficients ranged from 0.70 to 0.91 and from 0.40 to 0.98, respectively. DNA polymorphisms were highly consistent with phenotypic traits on rootstock C. maxima. The relationships between C. maxima from different origins were not clearly defined via morphological characteristics and RAPD analysis, suggesting some traits of C. maxima were specific to geographical location had disappeared or were weak in Heilongjiang province, which would not facilitate pumpkin breeding. Hybrid generations and their parent plants or sister lines were grouped into sub-clusters and showed little genetic distance according to both evaluation methods. Overall, morphological characteristics and RAPD markers were consistent and revealed high genetic diversity between C. maxima landraces from different origins.     

Genetic Diversity Among Brazilian Cultivars and Landraces of Tomato Lycopersicon Esculentum Mill. Revealed by RAPD Markers

Random amplified polymorphic DNA markers (RAPD) were used to estimate the variability of 35 tomato accessions (Lycopersicon esculentum Mill.). A total of 257 reproducibly scorable bands were obtained from 20 primers, 78.6% of which were polymorphic. The percentage distribution of RAPD markers shows a bimodal distribution, and the frequency of rare alleles is similar in commercial and landrace accessions. Genetic distances among accessions were calculated and a dendrogram showing the genetic relationships among them was constructed allowing for the separation of four groups. Twenty out of 23 Brazilian landraces fell within one group, whereas commercial cultivars were distributed in the four groups. AMOVA analysis of RAPD data showed that, despite the high within Brazilian landraces and commercial cultivars variation, these two groups are significantly different, indicating that landraces can be a source of variation for breeding programs.     

Genetic diversity in cowpea [Vigna unguiculata (L.) Walp.] as revealed by RAPD markers

The present study, using RAPD analysis, was undertaken to characterize genetic variation in domesticated cowpea and its wild progenitor, as well as their relationships. The materials used consisted of 26 domesticated accessions, including accessions from each of the five cultivar-group, and 30 wild/weedy accessions, including accessions from West, East and southern Africa. A total of 28 primers generated 202 RAPD bands. One hundred and eight bands were polymorphic among the domesticated compared to 181 among wild/weedy cowpea accessions. Wild accessions were more diverse in East Africa, which is the likely area of origin of V. unguiculata var. spontanea. Var. spontanea is supposed to have spread westward and southward, with a loss of variability, loss counterbalanceed in southern Africa by introgressions with local perennial subspecies. Although the variabilty of domesticated cowpea was the highest ever recorded, cultivar-groups were poorly resolved, and several results obtained with isozyme data were not confirmed here. However primitive cultivars were more diverse than evolved cultivars, which still suggests two consecutive bottlenecks within domesticated cowpea evolution. As isozymes and AFLP markers, although with a larger number of markers, RAPD data confirmed the single domestication hypothesis, the gap between wild and domesticated cowpea, and the widespread introgression phenomena between wild and domesticated cowpea.     

Genetic variability of Coffea arabica L. accessions from Ethiopia evaluated with RAPDs

The genetic diversity of 50 wild and semi-wild accessions of the Coffea arabica L. germplasm collection, gathered by the FAO and ORSTOM missions to Ethiopia, and maintained in Colombia by CENICAFE, was evaluated with RAPD markers. The evaluation was carried out in two phases: In phase one, the polymorphism of 8 Ethiopian accessions of different geographic origin, plus the cultivated variety 'Caturra' was assessed with the RAPD technique with forty-two 10-mer oligonucleotides. In phase two, 51 accessions were assessed with a set of 5 polymorphic primers that reproduced, with a correlation of 95%, the groups generated by the 24 polymorphic primers found in phase one. Principal Coordinate Analysis of molecular data revealed that a closely related group consisting of 86% of the Ethiopian C. arabica accessions evaluated are significantly different from the Caturra variety and could be used in a genetic breeding initiative to increase the variability of cultivated varieties. The results also indicate that a larger polymorphism is present in the Colombian replica of FAO Ethiopian coffee germplasm collection than previously reported.     

Microsatellites and RAPD Markers to Study Genetic Relationships Among Cowpea Breeding Lines and Local Varieties in Senegal

Genetic diversity in local cowpea varieties and breeding lines from Senegal were studied using random amplified polymorphic DNA (RAPD) and microsatellite (SSR) techniques. Among the 61 RAPD primers used, twelve show polymorphism. Fifteen of the 30 microsatellite primer pairs were polymorphic, detecting one to nine alleles per locus. The RAPD and SSR data were analyzed both separately and in combination to assess relationships among genetic lines. Although RAPD provided information on levels of genetic diversity, microsatellite markers are most effective in determining the relationship among cowpea accessions and varieties. The SSR results support the genetic diversification of cowpea in Senegal and underscore their potential in elucidating patterns of germplasm diversity of cowpea in Senegal. An erratum to this article is available at .     

Genetic variability evaluation in a Moroccan collection of barley, Hordeum vulgare L., by means of storage proteins and RAPDs

The genetic variation existing in a set of barley (Hordeum vulgare L.) landrace samples recently collected in Morocco was estimated. Two kinds of genetic markers, seed storage proteins (hordeins) and random amplified polymorphic DNA (RAPD), were used. Only six out of 31 landraces were subjected to RAPD analysis. Both kinds of markers, RAPD and storage proteins, yielded similar results, showing that the level of variation observed in Moroccan barley was high: all landraces showed variability; 808 different storage protein patterns (multilocus associations) were observed among 1897 individuals (2.32 seeds per association, on average) with an average of 43 multilocus associations per accession. In general, genetic variation within accessions was higher than between accessions. The 100 polymorphic RAPD bands generated by 21 effective primers were able to generate enough patterns to differentiate between uniform cultivars and even between individuals in variable accessions. One of the aims of this work was to compare the effectiveness of RAPD versus storage protein techniques in assessing the variability of genetic resource collections. On average hordeins were more polymorphic than RAPDs: they showed more alternatives per band on gels and a higher percentage of polymorphic bands, although RAPDs supply a higher number of bands. Although RAPD is an easy and standard technique, storage protein analysis is technically easier, cheaper and needs less sophisticated equipment. Thus, when resources are a limiting factor and considering the cost of consumables and work time, seed storage proteins must be the technique of choice for a first estimation of genetic variation in plant genetic resource collections.     

Relationships between an Italian Strawberry Ecotype and its Ancestor using RAPD Markers

Random-amplified polymorphic DNA (RAPD) markers were used to evaluate genetic variability among populations of an Italian strawberry ecotype, and to determinate genetic relationships between genotypes and their putative ancestor. A total of 65 selections and one cultivar ‘Madame Moutot’ (MM), were analysed to evaluate genetic variability present in Etna mountain area and to confirm as MM was one of the cultivars that originated the ecotype. A total of 222 RAPD markers was obtained using 16 decamer primers and 6 longer primers, 90.8% of the markers obtained by selected primers resulted polymorphic at least within analysed genotypes. RAPDs were used to calculate genetic similarity coefficients and to generate dendrograms representing genetic relationships among genotypes analysed. Cluster analysis displays as RAPD polymorphisms were able to characterize the genotype variability among closely related groups. The data show as MM could be considered the ancestral genotypes introduced in that area. The results obtained confirm that RAPD markers could be used as reliable markers to perform phylogenetic studies in Fragaria×ananassa Duch. ex Rozier. Giuseppe Bertino and Piero Spada - Coauthor involved in genotype selection and field management     

Genetic Characterization of Brazilian Annual <Emphasis Type="Italic">Arachis</Emphasis> Species from Sections <Emphasis Type="Italic">Arachis</Emphasis> and <Emphasis Type="Italic">Heteranthae</Emphasis> using RAPD Markers

The genus Arachis is divided into nine taxonomic sections. Section Arachis is composed of annual and perennial species, while section Heteranthae has only annual species. The objective of this study was to investigate the genetic relationships among 15 Brazilian annual accessions from Arachis and Heteranthae using RAPD markers. Twenty-seven primers were tested, of which nine produced unique fingerprintings for all the accessions studied. A total of 88 polymorphic fragments were scored and the number of fragments per primer varied from 6 to 17 with a mean of 9.8. Two specific markers were identified for species with 2n = 18 chromosomes. The phenogram derived from the RAPD data corroborated the morphological classification. The bootstrap analysis divided the genotypes into two significant clusters. The first cluster contained all the section Arachis species, and the accessions within it were grouped based upon the presence or absence of the ‘A’ pair and the number of chromosomes. The second cluster grouped all accessions belonging to section Heteranthae.     

Marker-assisted sampling of the cultivated Andean potato Solanum phureja collection using RAPD markers

The potato crop originated in the Andean highlands where numerous farmer's varieties and non-cultivated wild species exist. An Andean potato collection is held in trust at the International Potato Center (CIP) to preserve the biodiversity of this crop and ensure the supply of germplasm for potato improvement worldwide. A core collection representing the biodiversity of the Andean potato germplasm is under construction using morphological, molecular, and geographic data. One of the eight cultivated potato species, Solanum phureja, has been genotyped using the RAPD technique. A protocol suitable for large germplasm collection genotyping has been developed to process numerous samples at reasonable costs. From 106 RAPD primers evaluated, we have selected 12 primers yielding 102 polymorphic markers, which unambiguously discriminated all 128 accessions but 2 that are possible duplicates. The S. phureja germplasm collected throughout the Andean countries appears to have a homogeneous genetic constitution. There was no clear geographic pattern as indicated by cluster analysis of the RAPD data. A sub-group of 20 accessions has been identified on the basis of the marker data and selected to maximize molecular (RAPD) variance and polymorphism. The probability of capturing equal amounts of marker polymorphism in this sub-group of 20 accessions by random sampling is less than 40%. This set accessions represents our first group of accessions that may constitute a core of the S. phureja collection. This tentative core will be challenged for diversity content by alternate markers and agronomic traits. Hence, the methodology for sampling less than 10% of the base collection, proposed for core collections by Brown (1989), can be based on molecular marker data provided cost-efficient fingerprints are developed.     

Genetic Diversity of Traditional Chinese Mustard Crops Brassica juncea as Revealed by Phenotypic differences and RAPD Markers

This investigation was aimed at exploring the genetic diversity among nine typical accessions of Chinese mustard crops using random amplified polymorphic DNA (RAPD) markers and morphological comparison. Totally, 111 reproducible DNA bands were generated by 16 arbitrary primers, of which 91 bands were proved to be polymorphic. Based on pair-wise comparisons of the amplified bands, genetic similarities were obtained using Nei & Li's similarity coefficients and a dendrogram reflecting their relationships was made using the unweighted pair–group method with arithmetic averages (UPGMA). The result of cluster analysis indicated that the nine accessions were capable of being classified into two primary groups, one including accession 2 with expanded root (root mustard), accession 3 with entirely expanded whole stem (long-stem mustard), accession 6 with edible leaves (leaf mustard), accession 8 with edible seed stalk (seed stalk mustard) and another one including accession 4 with expanded basal stem (short-stem mustard), accession 5 with bulgy petiole (leafy bulgy mustard), and accession 9 with mustard-rich seed (seed mustard). Besides, accession 1 with expanded root (root mustard) and accession 7 with edible leaves and seed stalk (seed stalk mustard) were independent of other accessions in the dendrogram. Additionally, by cluster analysis based on highly reproducible RAPD markers, the accessions with similar edible parts of leaves or roots were not actually in the same phylogenetic groups. This implied that they were probably derived from different geographical origins with dissimilar genetic background and possessed higher genetic diversification. Furthermore, the results indicated that the traditional method for classifying Chinese mustard crops was not much reliable as it was largely dependent on phenotypic behaviors. Meanwhile, the phenotypic differences among individuals did not necessarily mean they must have sharp difference in genetic background as they met in the same group. Undoubtedly, these results aforementioned make this crop quite interesting to researchers for further investigating the molecular evolution of this special AABB group.     

Genetic variation and phylogenetic relationships among Indian Citrus taxa revealed by DAMD-PCR markers

Genetic variation and phylogenetic relationships among 50 wild and cultivated accessions of 19 Indian Citrus genotypes were examined through comparison of Directed Amplification of Minisatellite DNA (DAMD) markers and morphological characters. DAMD-PCR analysis with four primers resulted in amplification of a total of 45 bands, of which 35 (78 %) were polymorphic. Morphometric evaluation using 76 morphological characters showed high level of variability ranging from 0.18 to 1.00 (avg 0.39), whereas the Jaccard’s coefficient values of genetic similarity calculated from DAMD data ranged from 0.41 to 1.00 (avg 0.68), indicating moderate genetic divergence among the accessions studied. UPGMA dendrograms generated separately from morphometric and DAMD data segregated all the accessions of Citrus into four main clusters, each containing a true basic species and their probable hybrids. The grouping of individual accessions/genotypes under respective species or cultivars in DAMD dendrogram was based purely on their genetic relationships rather than geographical origin. There was no absolute congruence between the data and dendrograms generated from morphometric and DAMD analyses. The study demonstrates the resolving power of DAMD markers for discrimination of individual genotypes of Citrus under its respective species, hybrid or cultivar groups and inferring their genetic and phylogenetic relationships as well. This is the first report on application of DAMD markers in Citrus.